Anthocyanins are plants secondary metabolites important for plant adaptation to severe environments and potentially beneficial to human health. Purple colour of barley grain is caused by the pigments synthesized in pericarp. One or two genes determine the trait. One of them is Ant2 mapped on chromosome 2HL and is known to encode transcription factor (TF) with a bHLH domain. In plants, bHLH regulates anthocyanin biosynthesis together with TF harboring an R2R3-MYB domain. In wheat, the R2R3-MYBs responsible for purple colour of grain pericarp are encoded by the homoallelic series of the Pp-1 genes that were mapped on the short arms of chromosomes 7. In barley, in orthologous positions to wheat’s Pp-1, the Ant1 gene determining red colour of leaf sheath has been mapped. In the current study, we tested whether Ant1 has pleiotropic effect not only on leaf sheath colour but also on pericarp pigmentation. А set of near isogenic lines (NILs) carrying different combinations of alleles at the Ant1 and Ant2 loci was created using markers-assisted backcrossing approach. The dominant alleles of both the Ant1 and Ant2 genes are required for anthocyanin accumulation in pericarp. A qRT-PCR analysis of the Ant genes in lemma and pericarp of the NILs revealed that some reciprocal interaction occurs between the genes. Expression of each of the two genes was up-regulated in purple-grained line with dominant alleles at the both loci. The lines carrying dominant allele either in the Ant1 or in the Ant2 locus were characterized by the decreased level of expression of the dominant gene and scant activity of the recessive one. The Ant1 and Ant2 expression was barely detected in uncolored line with recessive alleles at both loci. The anthocyanin biosynthesis structural genes were differently regulated: Chs, Chi, F3h, Dfr were transcribed in all lines independently on allelic state of the Ant1 and Ant2 genes, whereas F3’h and Ans were activated in presence on dominant alleles of the both regulatory genes. The R2R3-MYB-encoding counterpart (Ant1) of the regulatory Ant2 gene was determined for the first time. The dominant alleles of both of them are required for activation of anthocyanin synthesis in barley lemma and pericarp. The R2R3-MYB + bHLH complex activates the synthesis via affecting expression of the F3’h and Ans structural genes. In addition, positive regulatory loop between Ant1 and Ant2 was detected. Earlier the interaction between the anthocyanin biosynthesis regulatory genes has been revealed in dicot plant species only. Our data demonstrated that the regulatory mechanism is considered to be more common for plant kingdom than it has been reported so far.

In wheat, anthocyanin pigments can be accumulated in pericarp tissues (under control of the Pp genes) resulting in purple-colored grain. In the current study, a strategy, based on the use of molecular and morphological markers, was applied to create purple-grained bread wheat cultivars adapted to the West Siberian region. The breeding scheme started from crossing of recipients (elite cultivars and lines) with donor lines carrying dominant alleles of the complementary genes Pp3 and Pp-D1. The F₂ hybrids passed three-step marker-assisted selection, and those having dominant Pp-D1Pp-D1Pp3Pp3 genotypes were backcrossed with the recurrent parents. The desired BC₁F_2-3 progenies were selected using morphological marker, while BC₁F₃ also passed through field evaluation. At this stage, 120 lines were selected and planted in individual 1 m² “breeding nursery (BN) plots” for assessment of heading dates, duration of vegetation period, resistance to powdery mildew, stem and leaf rusts, protein and gluten content, as well as productivity. After these investigations, a total of 17 promising anthocyanin-rich purple-grained lines characterized by multiple resistance and having best yield/quality characteristics were finally candidates for selection of commercial cultivars adapted to the West Siberian climate and suitable for functional food production.

Anthocyanins and melanins are phenolic pigments of plants and accumulate in seed envelopes of the barley grain, thereby giving them a blue, purple, or black color. To explore the effects of combined accumulation of anthocyanins and melanins in the grain, a barley near-isogenic line (NIL), characterized by simultaneous accumulation in both pigments, was developed using a marker-assisted approach. The presence of both pigments in the grain pericarp was evaluated by light microscopy. Emergence of anthocyanin pigmentation proved to be temporally separated from that of melanin, and the formation of anthocyanin pigments began at an earlier stage of spike maturation. During spike maturation, a significantly higher total anthocyanin content was noted in the created NIL than in the parental anthocyanin-accumulating NIL, indicating a positive influence of the Blp1 gene on the anthocyanin content at some developmental stages. In a comparative analysis of yield components, it was found that the observed differences between the barley NILs are possibly caused by environmental factors, and the presence of pigments does not decrease plant productivity. Our results should facilitate investigation into genetic mechanisms underlying overlaps in the biosynthesis of pigments and into breeding strategies aimed at the enrichment of barley varieties with polyphenols.

Breeding of cereals with anthocyanin-rich grains is promising for health food production. Here, we transferred genes responsible for activation of anthocyanin biosynthesis in the grain pericarp (Ant1 and Ant2) and aleurone (HvMyc2) to elite barley cultivars Aley, Tanay, and Vorsinsky-2 by marker-assisted backcrossing. Donors were Bowman lines BW648 and BW418. Three pairs of near-isogenic lines (NILs) with purple or blue colored grains were chosen in generation BC₆F₂ and propagated up to BC₆F₆. Genotyping-by-sequencing of resulting NILs and parental lines showed that the NILs carry 4.6–17.6% of donor SNPs including those at target loci. Unexpected big blocks of donor SNPs were revealed in some NILs on chromosomes 1H and 5H that do not carry target loci. The NILs possessed 274% and 12% higher anthocyanin (40.0–170.4 μg/g) and total phenolic content (2367.2–4396.8 μg/g), respectively, compared to original cultivars (18.3–33.1 μg/g and 2319.6–3768.5 μg/g). On average, antioxidant activity was 14% higher, although some lines showed reduced activity. NILs’ productivity depended on growth conditions and was similar to that of the recurrent cultivars. Hence, the applied breeding strategy is an effective approach to enrichment of grain anthocyanin content in barley.

The high adaptability of barley (Hordeum vulgare L.) facilitated its cultivation across almost the whole Russian territory and various extreme environments. However, the genetic diversity and population structure of historical Russian barley collections remain poorly characterized at the genomic level. This study provides the first comprehensive genomic description of the barley panel maintained in the GeneAgro seed bank. Using genotyping by sequencing (GBS), we obtained high-quality genotyping data of 158 Russian and 21 foreign accessions. We evaluated genome-wide diversity and population structure, revealing a clear genetic stratification into two-rowed and six-rowed barley. Russian accessions complemented the diversity of the more than 19,000 domesticated barley accessions from the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), expanding the representation of germplasm developed specifically for Russian environments and economic demands. To identify genomic regions that may have been shaped by breeding, we scanned the native GeneAgro barley accessions for selective footprints. We detected multiple such regions across the genome. Some were shared by two-rowed and six-rowed barley, while others were specific to one row type, which may reflect differences in their breeding histories. These regions contain candidate genes previously associated with spike architecture, plant morphology, flowering time, fertility, grain color, photosynthesis, seed dormancy and resistance to fungal pathogens. These candidates provide hypotheses for future allele-level and phenotypic studies.